Influenza viruses cause a highly contagious respiratory disease in humans. Influenza virus types A and B are distinguished by antigenic differences between their viral coat proteins. Influenza B viruses account for 30 - 40% of seasonal epidemics and are responsible for hundreds of thousands of deaths and countless loss of productivity worldwide each year. The long-term goal of this research program is to elucidate the mechanism(s) by which the influenza B non-structural protein 1 (NS1B) suppresses host innate immune response. One function of NS1B is to suppress interferon- (IFN-) synthesis in virus-infected cells. This activity has been mapped independently to each of its two domains, NS1B- NTD and NS1B-CTD. While there have been extensive structure-function studies of the NS1B-NTD, the mechanism by which the NS1B-CTD independently antagonizes activation of IFN expression is not known. Our laboratory has discovered that the NS1B-CTD is an RNA-binding domain. This totally unanticipated discovery is based on our 2.0- X-ray crystal structure of NS1B-CTD. Dimerization of NS1B-CTD creates a broad, strongly-conserved, basic surface, characteristic of nucleic-acid binding proteins. Fluorescence polarization (FP) studies demonstrate that NS1B-CTD binds RNA and (more weakly) DNA. Our specific hypothesis is that NS1B-CTD functions by binding dsRNA molecules, to suppress the innate immune response elicited by viral dsRNA. If this hypothesis is correct, disruption of these protein-nucleic acid interactions in virus-infected cells would provide new insights into the mechanisms of innate immune response, and provide the basis for the development of novel antiviral drugs and live attenuated vaccines. The aims of our R21 proposal are to (i) define the sequence and structural features of RNA required for binding to NS1B-CTD, and (ii) define the structural basis for molecular recognition between NS1B-CTD and RNA molecules. Biophysical methods, including FP spectroscopy, will be used to compare binding of NS1B-CTD to various synthetic RNA and DNA molecules. Ala scanning mutagenesis will be used to identify key protein residues contributing to RNA binding affinity. NMR, chemical crosslinking, amide hydrogen-deuterium exchange, and SAXS data will be combined to characterize the RNA-binding surface epitope of NS1B-CTD and to model the 3D structure of NS1B-CTD:RNA complexes. Efforts will also be made to determine 3D structures of these complexes by X-ray crystallography. The structural and biophysical information generated with this R21 Exploratory Research Grant will provide the starting point for a future multi-investigator R01 proposal directed to understanding the role and mechanism of NS1B-CTD in suppressing innate immune response in flu-infected cells.